CAR activation mechanism: What is unique about this system is the fact that xenobiotics do not directly bind to CAR to activate it. We have made the major breakthrough into deciphering this indirect activation mechanism, which has been a mystery for the past 10 years. Taking the previous finding that the protein phosphatase 2A inhibitor okadaic acid represses CAR activation by phenobarbital, we have been working on the hypothesis that CAR is inactivated by phosphorylation. Threonine 38 of human CAR (threonine 48 in mouse CAR) has now been identified as the primary site of PKC-mediated phosphorylation that inactivates CAR and sequesters it in the cytoplasm. Immuohistochemistry using an anti-phosphopeptide antibody showed that phenobarbital dephosphorylates threonine 38 to activate CAR and translocate it into the nucleus. Dephosphorylation of threonine 38, therefore, appears to be the primary mechanism for the indirect activation of CAR. This finding is currently revised for publication in Journal of Biological Chemistry. Xenobiotic-signal crosstalk mechanism: Upon activation by xenobiotics, CAR regulates genes differently from one another, conferring specificity to CAR-regulated gene expression. CAR acquires this specificity via crosstalk with these cellular signals: early response, growth and stress signals. We have identified early growth response 1 (EGR1) as the essential factor for CAR to activate the CYP2B gene. EGR l binds to multiple sites, looping the CYP2B6 promoter and enabling the distal CAR to interact with the proximal HNF4. We have also characterized the early response factor GADD45b (growth arrest and DNA-damage inducible 45bw as a new CAR co-activator. Target genes for CAR-mediated diseases: Chronic treatment with drugs (e.g. rifampicin, phenobarbtal and phenytoin) that activate CAR and/or PXR is known to cause bone diseases. Both CAR and PXR are found to interact with SMRT-VDR-VDRE within the CYP24A promoter, preventing dissociation of the co-repressor SMRT from the VDR and repressing VDR-dependent activation. In mouse, however, there is no such association between bone disease to the PXR-regulation of the CYP24A gene. Instead, we found that Pxr-/- mice develop hypophosphatemia and loss of bone mineral density. The Na/Pi-cotransporter SLC34A2 is severely down-regulated in the intestine of Pxr-/- mice, possibly causing hypophosphatemina. This hypophosphatemina study is currently revised for publication in Pharmacogenetics and Genomics.